J. Agric. Food Chem. 2005, 53, 549−553
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Study of 2-(2-Pyridyl)benzothiazoline as a Novel Fluorescent Probe for the Identification of Superoxide Anion Radicals and the Determination of Superoxide Dismutase Activity in Scallion Genus Foods LI ZHANG, BO TANG,*
AND
YI DING
College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, China
This paper presents a novel spectrofluorometric method using the novel fluorescent probe 2-(2pyridyl)benzothiazoline for the determination of superoxide dismutase (SOD) activity. The fluorescent probe was synthesized in house and fully characterized by elemental analysis and by IR and 1H NMR spectra. It could specially identify and trap superoxide anion radicals (O2•-), and then was oxidized by O2•- to form a strong fluorescence product. On the basis of this reaction, the spectrofluorometric method was proposed and successfully used to determine SOD activity. The proposed method has a better selectivity in the determination of reactive oxygen species, because the probe can be oxidized to afford a highly fluorescent product only by O2•- excluding hydrogen peroxide and hydroxyl radical. As a kind of simple, rapid, precise, and sensitive technique, it could avoid the errors caused by detection time and was applied to the measurement of SOD activity in scallion genus foods with satisfactory results. KEYWORDS: Superoxide anion radicals (O2•-); 2-(2-pyridyl)benzothiazoline (H. Py. Bzt); fluorescent probe; superoxide dismutase (SOD)
INTRODUCTION
Various free radicals are generated in the course of biological metabolism, such as superoxide anion radicals (O2•-), hydroxyl radicals (HO•), and ROO•, which are three typical ones (1). Free radicals, especially O2•-, have been considered extensively in myocardial ischemia and reperfusion injury in the past several years (2). Excessive O2•-, which is a toxic reactive oxygen, does great harm to photosynthetic pigment and membrane and further damages the photosynthetic systems. Moreover, O2•can be diverted into other more toxic radicals such as HO•, H2O2, and 1O2 (3), which can damage photosynthetic membranes and many biological molecules. In addition, O2•- can cause coronary, arteriosclerosis, and tumor diseases (4-6). For these reasons, eliminating O2•- from organisms is of great importance to prevent diseases and senescence. It is well-known that superoxide dismutase (SOD) is the scavenger of O2•- and an important indicator of the amount of O2•- in organisms. Therefore, establishing a precise, rapid, and sensitive method to determine SOD activity is of great applied value. At present, the common methods of determining SOD activity are autoxidation of pyrogallol (7) and hypoxanthinexanthine oxidase (HX-XO)-cytochrome reduction (8), both of which are spectrophotometric methods. In recent years, some new methods such as chemiluminescence (CL) (9), electron * Corresponding author (telephone +86-531-6180010; fax +86-5316180017; e-mail
[email protected]).
spin resonance (ESR) (10, 11), high-performance liquid chromatography (HPLC) (12), polarographic oxygen electrode (13), and immunity (14) have been developed. However, all of them have their disadvantages; for example, the instruments needed in ESR are too expensive, the operation in HPLC is complicated, the selectivity of CL is poor, and the sensitivity of spectrophotometry is low. Compared with the methods mentioned above, spectrofluorometry is simpler, more sensitive, and more selective. However, there are only a few papers on detecting SOD activity with spectrofluorometry so far. The other aspect wellknown is that the selectivity of reagents for reactive oxygen species (ROS) is important but difficult, because there are chain reactions of ROS in organisms, which result in the existence of many kinds of ROS. Commonly, O2•-, HO•, and H2O2 are the three main coexisting substances. In this work, a novel fluorescent probe, namely, 2-(2-pyridyl)benzothiazoline (H. Py. Bzt) synthesized in house, was applied to the identification of O2•- and the determination of SOD activity by spectrofluorometry. In the experiment, we found that only O2•- could oxidize H. Py. Bzt to form a strong fluorescence product, but H2O2 could not do so. Moreover, the same amount of hydroxyl radicals (HO•) also had no influence on the determination of O2•-, because it could not make the fluorescence intensity of the reaction system change at the measuring wavelengths. Therefore, the probe has a better selectivity. In addition, the reaction completed instantly, so the reaction time had no influence on the determination, which avoided the error
10.1021/jf049724a CCC: $30.25 © 2005 American Chemical Society Published on Web 01/11/2005
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Scheme 1. Synthesis of 2-(2-Pyridyl)benzothiazoline
caused by time. A comparison between the proposed method and the established method showed that the former had a satisfactory accuracy and good precision. The method was applied to determine SOD activity in garlic, scallion, and onion successfully. MATERIALS AND METHODS Apparatus. The fluorescence spectra and intensity were measured on a Cary Eclipse spectrofluorometer with a xenon lamp and 1.0 cm quartz cells (Varian). All pH measurements were made with a pH-3c digital pH-meter (Shanghai Lei Ci Device Works, China) with a combined glass-calomel electrode. The IR spectra were recorded on a PE-983 IR spectrometer (KBr disks cm-1, Perkin-Elmer, Norwalk, CT). Elemental analysis was performed on a PE-240 CHN elementary analytical meter (Perkin-Elmer). The 1H NMR spectra were recorded on an FX-90Q nuclear magnetic resonance spectrometer (DMSO as solvent, JEOL). The absorbance was recorded on a UV-265 spectrophotometer (Shimadzu). Reagents. × Fluorescent probe (1.00 × 10-4 mol L-1, synthesized in house) was prepared by dissolving an appropriate amount of 2-(2pyridyl)benzothiazoline in ethanol. The Na2S2O4 solution (6.80 × 10-3 mol L-1) was prepared by dissolving an appropriate amount of Na2S2O4 in 0.10 mol L-1 NaOH. A stock solution of SOD (1.00 × 10-2 g L-1) was prepared in water (stored in a refrigerator). A solution of H2O2 (3.40 × 10-4 mol L-1) was standardized by titration with potassium permanganate. The following solutions were prepared in doubly distilled water: pyrogallol, 3.00 × 10-3 mol L-1; Na2B2O7-NaOH (pH 11.60, 0.10 mol L-1) buffer solution, and Tris-HCl (pH 8.20, 0.10 mol L-1) buffer solution. 2-Pyridinaldehyde and o-aminobenzenothiol were purchased from Fluka. All chemicals used were of analytical reagent grade, and doubly distilled water was used throughout. Synthesis and Properties of H. Py. Bzt. 2-(2-Pyridyl)benzothiazoline was prepared by condensation of the appropriate 2-pyridinaldehyde (0.030 mol) with o-aminobenzenothiol in a 1:1 molar ratio in benzene (15). The synthesis reaction is shown in Scheme 1. The mixed solution was heated under reflux for ∼2-3 h. After the solvent was removed under reduced pressure, the yellow crude solid was obtained. Then it was recrystallized from benzene and dried in a vacuum. Melting point: 81-81.5 °C; 1H NMR (90 MHz, DMSO-d6, 25 °C, TMS) δ 6.3 (s, 1H, C-H), 4.3 (s, 1H, N-H), 7.2-8.5 (4H, pyridine-H), 6.6-7.2 (4H, benzol-H); IR (KBr pellet) ν (cm-1) 3250 (NsH), 3105 (CsH), 1595 (CdC). Elemental analysis (%) calcd for C12H10N2S (found): C, 67.24 (67.31); H, 4.70 (4.81); N 13.70 (13.60). The data were in agreement with the values reported in refs 15 and 16. Experimental Procedure. Determination of O2•- and ScaVenging Effect of O2•- by Standard SOD. Into a 10-mL graduated color comparison tube were added 0.50 mL of Na2S2O4 (6.80 × 10-3 mol L-1), 1.50 mL of Na2B4O7-NaOH buffer solution (pH 11.60, 0.10 mol L-1), and 3.00 mL of H. Py. Bzt in turn. Then it was diluted to volume with doubly distilled water and equilibrated. The fluorescence intensity was measured at λex/em ) 377/528 nm against a reagent blank. The excitation and emission slits were both set to 5 nm. A certain amount of standard SOD solution (0.20, 0.40, 0.60, 0.80, and 1.00 mL, respectively) was added additionally. The fluorescence intensity with SOD and Na2S2O4 was recorded as Fs, only with Na2S2O4 was F, without SOD or Na2S2O4 was F0. Then the scavenging percentage (P) of SOD to superoxide anion radicals was calculated by P ) (F - Fs)/(F - F0) × 100%. Determination of the ActiVity of SOD Extracted from Samples. In actual sample analysis, 0.20 mL of SOD extracted from scallion genus foods was added additionally and the reagent blank was carried out at the same time. The SOD amount with the P at 50% was assumed as a unit, and then the calculation formula of SOD activity was as follows: SOD activity (U mL-1) ) PVTn/(50%VM), where VT (mL) is the total
Figure 1. Excitation and emission spectra: 1, Na2S2O4 + buffer + H. Py. Bzt; 2, buffer + H. Py. Bzt; 3, H2O2 + buffer + H. Py. Bzt; 4, HO• [obtained from H2O2 (3.40 × 10-4 mol L-1) + Co2+] + H. Py. Bzt; 5, buffer + Na2S2O4 (3.40 × 10-4 mol L-1), H. Py. Bzt (3.00 × 10-5 mol L-1), buffer solution (pH 11.60, Na2B4O7−NaOH), H2O2 (0.003%), Co2+ (5.00 × 10-5 mol L-1). volume of the solution to be determined, VM is the volume of the SOD extract, and n is the dilution fold of the SOD extract (VT/VM). Then the SOD activity unit could be converted into units per gram according to the SOD content in the samples. Extract of SOD in the Samples. Garlic, scallion, and onion tegument and root were removed and dried in air; 1.0000 g of each sample and 2.00 mL of phosphate buffer solution (pH 7.80, 0.050 mol L-1) were mixed together. Having been frozen for 2 h, they were pounded into a paste and then diluted to 8.00 mL, frozen for 1 h continuously, and centrifuged (4000 rpm) for 15 min (17). After that, 5.40 mL of supernatants was transferred into three centrifuge tubes, and then cold ethanol (0.50 mL) and CHCl3 (0.50 mL) were added. After being equilibrated, they were centrifuged for 5 min. The supernatant was the SOD extract and stored in a refrigerator. Determination of SOD ActiVity by Classic Autoxidation of Pyrogallol. Into a 10-mL color comparison tube were added 5.00 mL of Tris-HCl (pH 8.20) buffer solution, 0.30 mL of pyrogallol (3.00 × 10-3 mol L-1), and 0.20 mL of SOD extract in turn. The mixture was diluted to volume with doubly distilled water. The solution was equilibrated, and its absorbance was measured at 320 nm against a reagent blank (7). The amount of SOD that could restrain the autoxidation rate of pyrogallol to 50% min-1 mL-1 at 25 °C was defined a SOD activity unit (units mL-1). The calculation formula of SOD activity is as follows: SOD activity (units mL-1) ) [(Vp - Vs)/(Vp × 0.5)] × VTn/Vs, where Vp is the autoxidation rate of pyrogallol, Vs is the autoxidation rate of samples, VT (mL) is the total volume of the solution to be determined, and n is the dilution fold of the SOD extract. Then the SOD activity unit could be converted into units per gram according to the SOD content in the samples. RESULTS AND DISCUSSION
Mechanism of the Determination. In this study, O2•- was produced by the reaction S2O42- + O2 + 4HO- ) 2SO32- + 2H2O + O2•- (18), and the reaction equilibrium constant (K) and conditional reaction equilibrium (K′) were 7.6 × 1018 and 7.6 × 108, respectively, which were calculated according to the standard and conditional redox potentials of SO32--S2O42- and O2-O2•- (19). Therefore, the reaction could take place spontaneously and completely in the alkaline solution saturated by O2, and the concentration of O2•- could remain stable for at least 1.5 h in 0.10 mol L-1 of NaOH. Because H. Py. Bzt could react with O2•- to form a strong fluorescence product, the amount of O2•- was determined indirectly by measuring the changes of fluorescence intensity. According to the proposed method, the excitation and emission spectra were recorded (Figure 1) and the fluorescence intensity was measured at λex/em ) 377/528 nm. It could be found that
Novel Fluorescent Probe for Identification of Superoxide Anion Radicals
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Figure 2. Autoxidation of pyrogallol: 1, pyrogallol + buffer + H. Py. Bzt; 2, buffer + H. Py. Bzt; 3, pyrogallol + buffer pyrogallol (3.40 × 10-4 mol L-1), H. Py. Bzt (3.00 × 10-5 mol L-1), buffer solution (pH 8.20, Tris-HCl). Scheme 2
H. Py. Bzt itself had no fluorescence and the fluorescence intensity increased obviously after the oxidation reaction took place. However, H2O2 could not oxidize the probe [Figure 1(3)], and the same amount of hydroxyl radical (HO•) could not make its fluorescence intensity change at the measuring wavelengths [Figure 1(4)]. Although a 10-fold amount of HO• can make the fluorescence intensity increase, in real samples HO• is derived from O2•- and its life is only 10-4 s with a very low concentration of 10-8 mol L-1 (20), so the amount of HO• existing in biology cannot reach the amount of O2•-. In addition, the reaction of H. Py. Bzt trapping O2•- was sensitive and rapid. Therefore, HO• could not interfere with the determination of O2•- in real samples. The probe could identify O2•- especially, and the proposed method has a better selectivity in the determination of ROS. To investigate the reaction mechanism, the oxidization product was synthesized; 0.050 g of H. Py. Bzt was dissolved in 10 mL of ethanol, 2.00 g of Na2S2O4 was dissolved in 10 mL of NaOH (0.10 mol L-1), they were mixed together, and 20 mL of buffer solution was added. Then the solvent was removed, and the residue was extracted by dichloroethane. The crude product was obtained as a green solid. The pure brown product was obtained by recrystallization in ethanol: melting point, 131-132 °C; 1H NMR (90 MHz, DMSO-d6, 25 °C, TMS) δ 7.6-8.6 (4H, pyridine-H), 7.3-7.6 (4H, benzol-H); IR (KBr pellet) ν (cm-1) 1637 (CdN), 3105 (CsH), 1595 (CdC). Elemental analysis (%) calcd for C12H8N2S (found): C, 67.90 (67.92); H, 3.75 (3.77); N, 13.26 (13.21). As shown in 1H NMR spectra, peaks corresponding to N-H (4.3) and C-H (6.3) of H. Py. Bzt disappeared and the other signals moved to a lower field, which indicated that the product had a larger conjugated system. This also could explain the fluorescence intensity increase. In the IR spectrum, NsH (3250 cm-1) and CsH (3105 cm-1) in H. Py. Bzt disappeared and the CdN absorption band at 1673 cm-1 appeared. All spectral data were consistent with structure II in Scheme 2. The structure was further confirmed by elemental analysis. Therefore, the mechanism could be presumed as given in Scheme 2. O2•- oxidized the probe by
deleting hydrogen to yield the compound 2-(2-pyridyl)benzothiazol (Scheme 2, II), which had better rigidity and a larger conjugated system (free electron pairs on S also conjugating with phenyl ring). Due to its larger conjugated system, the fluorescence intensity increased obviously after the probe reacted with O2•-. To confirm that it was O2•- obtained in alkaline Na2S2O4 that made the fluorescence increase, several related compounds containing sulfur were tested. It was found that neither Na2SO4, Na2SO3, nor Na2S could make the fluorescence intensity increase. Neutral and alkaline solutions of Na2S2O4 reacting with H. Py. Bzt were also tested, and the results showed that the fluorescence intensity did not change in the neutral water solution, but it increased obviously in the alkaline solution. This could be taken as evidence that only O2•- obtained in alkaline Na2S2O4 solution could oxidize the probe and make the fluorescence intensity increase. To validate the conclusion further, pyrogallol was applied as the source of O2•- to react with the probe. The results were the same as those with alkaline Na2S2O4 (Figure 2), and the fluorescence intensity increased with longer time due to the slow autoxidation rate of pyrogallol. This demonstrated that there existed O2•- in alkaline Na2S2O4 solution, which also could be confirmed from the experiment of SOD scavenging O2•-. Compared with autoxidation of pyrogallol as the source of O2•-, alkaline Na2S2O4 had a rapid rate, so it was unnecessary to consider the influence of time on determination. This not only avoided experimental error but also improved determination speed. Optimization of Experimental Variables. Effect of pH and Amount of Buffer Solution. As shown in Figure 3a, the experiment showed that the optimal pH for producing O2•- was in the range of 11.00-11.80. In comparison, Na2B4O7-NaOH was more sensitive within NH4Ac-NaOH, NH4Cl-NH3‚H2O, and Tris-HCl buffer solution systems. Therefore, Na2B4O7NaOH buffer solution was selected. The relative fluorescence intensity (∆F) of the system was high and stable when the amount of buffer solution was from 1.00 to 2.00 mL (Figure 3b). Thus, 1.50 mL of Na2B4O7-NaOH (0.10 mol L-1) buffer solution (pH 11.60) was chosen throughout the experiment. Effect of Reaction Time. Different reaction times were tested. The results showed that the ∆F did not change with different lay-side time (Figure 4). This indicated that the reaction was rapid; it could react completely in an instant due to its rapid reaction rate, so time had no influence on the determination
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Figure 5. Effect of Na2S2O4. Table 1. Effect of Addition Order of Reagents addition sequence of reagent
∆F (au)
(1) buffer + H. Py. Bzt + Na2S2O4 (2) buffer + Na2S2O4 + H. Py. Bzt (3) H. Py. Bzt + Na2S2O4 + buffer (4) H. Py. Bzt + buffer + Na2S2O4 (5) Na2S2O4 + H. Py. Bzt + buffer (6) Na2S2O4 + buffer + H. Py. Bzt
150 230 163 171 89 268
Table 2. Effect of Organic Solvents
Figure 3. Effect of pH (a) and amount of buffer solution (b).
Figure 4. Effect of reaction time.
and did not need to be considered. This not only improved the determination rate but also minimized the errors caused by detection time. Effect of Concentration of Na2S2O4. The concentration of Na2S2O4 determined the yield of O2•-. The experimental results showed that ∆F had a linear relationship with Na2S2O4 in the range from 0.00 to 3.20 × 10-4 mol L-1, and then remained high and stable when the concentration of Na2S2O4 was between 3.20 × 10-4 and 3.60 × 10-4 mol L-1 (Figure 5). Therefore, 3.40 × 10-4 mol L-1 of Na2S2O4 was selected throughout the experiment. Effect of the Concentration of Fluorescent Probe. Because H. Py. Bzt was the trapper of O2•-, its concentration directly determined whether O2•- was trapped completely, which decided the precision and sensitivity of the method. ∆F increased with the increase of the trapper concentration and then remained stable when it was between 2.50 × 10-5 and 3.50 × 10-5 mol L-1. Therefore, 3.0 × 10-5 mol L-1 of the probe was selected.
au
methanol
ethanol
2-propanol
acetonitrile
DMF
acetone
water
F F0 ∆F
40 138 98
33 137 134
43 133 90
39 194 155
61 175 114
42 149 107
13 214 201
Effect of Addition Sequence of Reagents. The addition sequence of reagents is detailed in Table 1. The results indicated the sixth one was the best. The reason was that Na2S2O4 in alkaline solution could yield more O2•-. Effect of Organic SolVents. Two milliliters of different organic solvents was added additionally, and then the fluorescence intensity was measured according to the experimental method above. The results are shown in Table 2. It can be seen that ∆F of the system without any organic solvent was highest. Therefore, water but not organic solvent was selected to dilute to volume. Reproducibility of the Method. According to the experimental method, 11 determinations were made every other day. The standard deviation was 1.25, and the relative standard deviation of the methods was 0.46%, which showed that the reproducibility of the proposed method was very good. Effect of Interferences. Thirty-one interferences (common components in organisms) were studied individually to investigate their effects on the determination of O2•- obtained from 3.4 × 10-4 mol L-1 of Na2S2O4 by the procedure. An error of (5% in the relative fluorescence intensity was considered to be tolerable. No interference was encountered from (tolerable ration in moles) Na+, Cl- (over 1500); glucose, lactin (1000); sucrose, K+, Mg2+ (500); Cu2+, Ca2+, Br-, NO3- (200), lactose, SO42-, I- (120); Zn2+ (100); L-phenylalanine, Co2-, Pb2+ (80); HAS, thymine, cytosine (40); adenine, guanine (30); thiourea, DNA (20); RNA, BSA (15); tryptophan, DL-tyrosine (10); and Fe3+, Al3+ (2). The experimental results showed that there was no interference for most biological components. Scavenging Effect of O2•- by Standard SOD. SOD is the specific scavenger of O2•-; its scavenging effect on O2•- could
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Figure 6. Relationship between SOD and scavenging percentage. Table 3. Determination of SOD Activity in Samples x¯ ± SD (units g-1) sample
no.
proposed method
standard method (7)
garlic scallion onion
7 7 7
184.2 ± 5.2 70.5 ± 4.5 50.3 ± 4.8
182.1 ± 5.6 72.2 ± 5.2 51.2 ± 6.0
be used to verify the efficiency of the proposed method. The experimental results showed that there was a good linear correlation (R ) 0.9995) between scavenging percent and SOD quantity (Figure 6). The calibration equation was ∆F ) 82.02 + 0.995C (C is in 10-4 g L-1). Therefore, the proposed method was effective. Determination of SOD Activity in Samples. On the basis of the proposed experimental method, SOD activity in garlic, scallion, and onion was determined successfully. The results obtained were compared with the autoxidation of pyrogallol method (Table 3). The former results were similar to the latter ones, which proved that the proposed method was efficient and useful. Compared with the classical method, the proposed method had a higher sensitivity and more rapid detection rate, because the autoxidation rate of pyrogallol was determined, then the detection time had to be controlled strictly in the classical method, and each rate determination needed at least 25 min, whereas the proposed method needed to measure only the fluorescence intensity, which could be recorded at any time after mixing without considering time, and each determination was completed in 1 min. The autoxidation of pyrogallol method had a close relationship with the detection time, which made the determination discommodious and caused bigger errors, and metal ions had more interference on the classical method. ABBREVIATIONS USED
O2•-, superoxide anion radicals; H. Py. Bzt, 2-(2-pyridyl)benzothiazoline; SOD, superoxide dismutase; HO•, hydroxyl radicals. LITERATURE CITED (1) Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and Medicine; Clarendon Press: Oxford, U.K., 1985; pp 11-14. (2) Lowe, R. D.; Snook, R. D. Sensitive spectrohotometric determination of aluminum using thermal lens spectrometry. Anal. Chim. Acta 1991, 250, 95-104.
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